# Reconstituting heterochromatin and gene silencing in vivo

> **NIH NIH F32** · HARVARD MEDICAL SCHOOL · 2020 · $15,501

## Abstract

PROJECT SUMMARY
Heterochromatin plays critical roles in maintaining genome stability and transcriptional gene silencing (TGS). It
has become increasingly clear that misregulation of pathways influencing heterochromatin integrity cause or
contribute to many human maladies, including numerous cancers. The composition and function of
heterochromatin domains are largely conserved from the fission yeast Schizosaccharomyces pombe to
humans and other metazoans. Heterochromatin establishment, epigenetic maintenance, and TGS in these
organisms are complex processes regulated by numerous chromatin-associated factors. Despite such
complexity, core principles of heterochromatin biology have been proposed but remain speculative. This study
will experimentally test and articulate these core principles by distilling essential features of heterochromatin in
a highly-controlled and orthogonal environment. This research proposal is composed of two aims. Aim 1 is to
reconstitute histone 3 lysine 9 methylation (H3K9me)-dependent heterochromatin in Saccharomyces
cerevisiae cells, which naturally lack H3K9me, with the goal of defining the minimal requirements for a
repressive and heritable chromatin state conserved from fission yeast to human. Successful construction of an
H3K9me-dependent heterochromatin domain in S. cerevisiae cells will provide a unique system for
investigating how heterochromatin is epigenetically inherited and how it silences transcription and limits other
DNA transactions. Accordingly, Aim 2 is to utilize in vivo reconstituted H3K9me-dependent heterochromatin to
investigate the mechanism of TGS. H3K9me-dependent heterochromatin will be reconstituted in S. cerevisiae
cells by sequentially recruiting S. pombe and human heterochromatin-associated proteins to a specific S.
cerevisiae genomic locus. This minimal heterochromatin domain will then be utilized to test three models of
TGS and assess the contribution of histone deacetylation and nucleosome remodeling to the formation of silent
chromatin domains. A combination of synthetic biology-, next generation sequencing-, and proteomics-based
experimental approaches will be utilized to execute this research plan. This work has potential to transform our
mechanistic understanding of heterochromatin formation and thereby inform future studies aimed at reversing
defects in heterochromatin-associated processes underlying human diseases. Furthermore, the reconstitution
of heterologous heterochromatin domains in vivo with factors found in S. pombe and human cells will facilitate
the development of cell-based assays amenable to high-throughput screens for small molecules that modulate
the function of proteins and protein complexes involved in heterochromatin formation and disease progression.
The proposed studies will thus deepen our understanding of fundamental processes underlying human
diseases and open new avenues to their treatment.

## Key facts

- **NIH application ID:** 9896664
- **Project number:** 5F32GM131438-02
- **Recipient organization:** HARVARD MEDICAL SCHOOL
- **Principal Investigator:** Andy Yuan
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $15,501
- **Award type:** 5
- **Project period:** 2019-02-01 → 2020-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9896664

## Citation

> US National Institutes of Health, RePORTER application 9896664, Reconstituting heterochromatin and gene silencing in vivo (5F32GM131438-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9896664. Licensed CC0.

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